Cell culture media extending materials and methods

ABSTRACT

This disclosure provides a cell culture media extending material capable of releasing nutrients into the cell culture environment slowly over time. In embodiments, this material is a part of a cell culture vessel. In embodiments, the material is a coating or a film on a surface of a cell culture material. In additional embodiments, the material is a surface upon which cells are cultured, such as a cell culture vessel or a microcarrier.

This application claims the benefit of priority under 35 U.S.C. §119 ofU.S. Provisional Application Ser. No. 62/084,356 filed on Nov. 25, 2014the content of which is relied upon and incorporated herein by referencein its entirety.

BACKGROUND

In cell culture systems, nutrients that cells require to survive inculture are present in liquid media that bathes cells. To maintain cellsin static culture for a long period of time, concentrations of nutrientssufficient to feed cells over that long period of time are provided inthe media. However, nutrients break down over time. For example,glutamine, an essential amino acid, and glucose, necessary for energymetabolism, are provided in cell culture media. These nutrients degradeover time. The breakdown of these nutrients can be affected by theconcentration of the nutrients themselves. Breakdown products ofnutrients such as glutamine and glucose can be toxic to cells.Therefore, providing relatively high concentrations of nutrients in cellculture media in order to extend the life of cell culture may result inmedia which becomes toxic to cells as nutrients break down over time.These effects may limit the lifespan of cell culture.

The disclosure relates to materials useful for the sustained andcontrolled delivery of nutrients to cultured cells in order to provideenvironments suitable for extending the duration of cell culture.

SUMMARY

The disclosure provides a surface, exposed to cell culture, having mediaextending materials which have sequestered or captured cell culturenutrients and provide a mechanism for the release of nutrients in acontrolled and sustained way which support cells in culture for a longertime. In embodiments the media extending materials are coatings orfilms, applied to a cell culture surface, which are capable of releasingcell culture nutrients over time. In embodiments the media extendingmaterials disclosed herein are polymers which swell to less than 50%water by weight in an aqueous environment, which are mixed withnutrients, or have captured nutrients, or contain nutrients. Inembodiments, nutrients are captured or sequestered in polymer incrystalline or solid form.

In additional embodiments, the disclosure provides methods of culturingcells in the presence of a cell culture surface having media extendingmaterials where the nutrients, captured in a polymer matrix, dissolve inthe presence of aqueous media, and are slowly released into the media.When the nutrients dissolve in an aqueous environment, an osmotic pumpmay be created in pockets in the polymer matrix, allowing nutrients toflow from the more concentrated environment of the polymer pocket, intothe media. This sequestration of nutrients in the polymer matrix allowsfor the slow introduction of nutrients into media without opening a cellculture vessel. This sequestration also allows cells to be cultured, instatic culture or in dynamic culture conditions, in the presence oflower concentrations of nutrients which, in higher concentrations, maybe toxic to cells, directly or indirectly.

In embodiments, the disclosure provides a cell culture vessel having asurface comprising a cell culture media extending material. Inembodiments, the cell culture extending material can be a coating or afilm, or a coating which is a film. In embodiments, the cell culturemedia extending material comprises a hygroscopic polymer comprising atleast one sequestered nutrient. In embodiments, the cell culture vesselcomprises at least one cell culture compartment, each cell culturecompartment comprising a bed, a ceiling and at least one wall. Inembodiments the cell culture media extending material is on a bed, aceiling or a wall of a cell culture compartment, or a combination of thebed, the ceiling or the wall.

In embodiments, the disclosure provides a cell culture media extendingmaterial comprising a hygroscopic polymer and at least one nutrient. Inembodiments, the cell culture media extending material forms part of acell culture vessel or part of a microcarrier. In embodiments, thehygroscopic polymer comprises EVA and the at least one nutrient isglucose, glutamine or a combination of glucose and glutamine.

In embodiments, the disclosure provides a method of culturing cellsincluding: (a) introducing cells into a cell culture chamber comprisinga cell culture media extending material, wherein the cell culture mediaextending material comprises a hygroscopic polymer comprising at leastone sequestered nutrient; (b) incubating the cells in static culture inthe presence of media having an L-glutamine concentration less than 2mM. In embodiments, the glutamine concentration is between 0 and 1.5 mM.

BRIEF DESCRIPTION OF THE DRAWINGS

In embodiments of the disclosure:

FIGS. 1a and 1b are illustrations of a cell culture vessel in anembodiment. FIG. 1b is a cross-section of the cell culture vessel shownin FIG. 1 a, taken at plane A-A shown in FIG. 1 a.

FIG. 2 is an illustration of a cell culture surface in the form of amicrocarrier, in another embodiment.

FIGS. 3a-3e are cross-sections of a cell culture vessel taken at planeA-A shown in FIG. 1 b, showing embodiments of the material on interiorsurfaces of a cell culture vessel.

FIGS. 4a and 4b are images of embodiments of cell culture extendingmaterials, in the absence (FIG. 4a ) and in the presence (FIG. 4b ) ofdPBS.

FIG. 5 is a partial cut-away illustration of a multi-layer cell culturevessel, in another embodiment.

FIG. 6 is a cross-section of a cell culture vessel, taken at plane B-Bshown in FIG. 5, showing five (5) embodiments of the material oninterior surfaces of a multi-layer cell culture vessel.

FIG. 7 is an illustration of a method of using the material, in anembodiment.

FIGS. 8a-8d are illustrations of embodiments of methods of using fourembodiments of the material in a multi-layer flask environment.

FIG. 9 is a graph showing glucose consumption vs. ammonium production inthe presence of traditional media having a 4 mM L-glutamineconcentration (and a 1 g/L glucose concentration) compared toexperimental low glutamine media conditions having a 0.5 mM L-glutamineconcentration (and a 1 g/L glucose concentration).

FIG. 10 is a graph illustrating CHOk1 cells harvested after 96 hoursgrowing in traditional media having a 4 mM L-glutamine concentration(and a 1 g/L glucose concentration) compared to experimental lowglutamine media conditions having a 0.5 mM L-glutamine concentration(and a 1 g/L glucose concentration).

FIG. 11 is a graph illustrating ammonium production per cell per dayafter 96 hours growing in traditional media having a 4 mM L-glutamineconcentration (and a 1 g/L glucose concentration) compared toexperimental low glutamine media conditions having a 0.5 mM L-glutamineconcentration (and a 1 g/L glucose concentration).

FIG. 12 is a graph illustrating glucose release in mg/cm² from glucoseand glutamine-containing embodiments of the material over time.

FIG. 13 is a graph illustrating glutamine release in mg/cm² from glucoseand glutamine-containing embodiments of the material over time.

FIG. 14 is a graph illustrating glucose release in g/L fromglucose-containing embodiments of the material over time.

FIG. 15 is a graph illustrating glucose release in g/L from aglucose-containing embodiment of the material over time.

FIG. 16 is a graph illustrating glucose release in g/L from anotherglucose-containing embodiment of the material over time.

FIG. 17 is a graph illustrating glucose release in g/L from anotherglucose-containing embodiment of the material over time.

FIG. 18 is a graph illustrating glucose release in g/L from anotherglucose-containing embodiment of the material over time.

DETAILED DESCRIPTION

In embodiments, this disclosure provides a cell culture media extendingmaterial capable of releasing nutrients into a cell culture environmentslowly over time. In additional embodiments, the material is exposed tocell culture media. In embodiments, this material is a part of a cellculture vessel. In embodiments, the material is a coating or a film on asurface of a cell culture material. In additional embodiments, thematerial is a surface upon which cells are cultured, such as a cellculture vessel or a microcarrier.

In embodiments, the disclosed cell culture media extending material, andthe disclosed method of making and using the cell culture mediaextending material, provide one or more advantageous features oraspects, including for example those discussed below. Features oraspects recited in any of the claims are generally applicable to allfacets of the invention. Any recited single or multiple feature oraspect in any one claim can be combined or permuted with any otherrecited feature or aspect in any other claim or claims.

Definitions

“Nutrient” refers to any compound or component, whether of chemical orbiological origin, that can be used in the disclosed material, which iscontacted with cell culture media to maintain or promote the growth orproliferation of cells, or cellular production of biologically activesubstances. “Component,” “nutrient,” “sustenant,” or “ingredient” can beused interchangeably and all refer to such compounds. Nutrients that canbe used in cell culture media can include, for example, amino acids,salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fattyacids, proteins, and like substances, or combinations thereof. Otheringredients that can promote or maintain cultivation of cells in vitro(e.g., in cell culture) can be selected by those of skill in the art, inaccordance with a particular need.

“Material” means the cell culture media extending material disclosedherein, in embodiments.

“Sequestered” means a nutrient, in solid or crystalline form, that iscaptured in or mixed in a polymer matrix. Nutrients that are trappedbeneath a layer of polymer or trapped between layers of polymer are also“sequestered”.

“Include,” “includes,” or like terms means encompassing but not limitedto, that is, inclusive and not exclusive.

“About” modifying, for example, the quantity of an ingredient in acomposition, concentrations, volumes, process temperature, process time,yields, flow rates, pressures, viscosities, and like values, and rangesthereof, or a dimension of a component, and like values, and rangesthereof, employed in describing the embodiments of the disclosure,refers to variation in the numerical quantity that can occur, forexample: through typical measuring and handling procedures used forpreparing materials, compositions, composites, concentrates, componentparts, articles of manufacture, or use formulations; through inadvertenterror in these procedures; through differences in the manufacture,source, or purity of starting materials or ingredients used to carry outthe methods; and like considerations. The term “about” also encompassesamounts that differ due to aging of a composition or formulation with aparticular initial concentration or mixture, and amounts that differ dueto mixing or processing a composition or formulation with a particularinitial concentration or mixture.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The material, and the method of making and using the material of thedisclosure can include the components or steps listed in the claims,plus other components or steps that do not materially affect the basicand novel properties of the materials, or methods of making and use,such as a particular apparatus or vessel configuration, particularadditives or ingredients, a particular agent, a particular structuralmaterial or component, a particular incubation or culture condition, orlike structure, material, or process variable selected.

The indefinite article “a” or “an” and its corresponding definitearticle “the” as used herein means at least one, or one or more, unlessspecified otherwise.

Abbreviations, which are well known to one of ordinary skill in the art,may be used (e.g., “h” or “hrs” for hour or hours, “g” or “gm” forgram(s), “mL” for milliliters, and “rt” for room temperature, “nm” fornanometers, and like abbreviations).

Specific and preferred values disclosed for compositions, ingredients,additives, dimensions, conditions, and like aspects, and ranges thereof,are for illustration only; they do not exclude other defined values orother values within defined ranges. The compositions and methods of thedisclosure can include any value or any combination of the values,specific values, more specific values, and preferred values describedherein, including explicit or implicit intermediate values and ranges.

Successful culturing of cells requires consideration of nutrientconsumption and metabolic waste accumulation. Cells in culture typicallygrow in an aqueous media environment. Cells can be grown in staticculture, in perfusion culture, or in mixed or shaken culture. Cellscultured in static culture conditions, conditions that do not have aconstant flow of nutrient media in or through the cell culture vessel,sit in cell culture media for extended periods of time. Nutrients,provided by the cell culture media, are used up by the cultured cellsover time, and byproducts, or waste products, build up. These wasteproducts may be toxic.

Cell culture media contains vital amino acids, sugars, salts, growthfactors, and other ingredients important for maintaining the health ofcells in culture. One important amino acid ingredient in cell culturemedia is glutamine. Glutamine decomposes spontaneously in cell culturemedia over time. Cell culture medium is normally formulated withexcessive glutamine to compensate for its decomposition. However,ammonium is produced by to the spontaneous decomposition of L-glutamine.Ammonium is also produced as a result of cell metabolism. Too muchammonium in cell culture media may be toxic to cells in culture. It hasbeen demonstrated that fed-batch systems benefit from use of lowsustained glutamine concentration to reduce the rate of ammoniumformation. This has been achieved via frequent or continuous feedingwhich increases the volume of media required and dilutes cultureproducts formed.

Cell culture media can be, for example, aqueous-based and can comprise anumber of ingredients in a solution of, for example, deionized,distilled water to form a “basal media.” Any basal medium can be used inaccordance with the disclosed methods. The basal media can include, forexample, one or more of the following ingredients: amino acids,vitamins, organic salts, inorganic salts, trace elements, bufferingsalts, and sugars. Preferably, the basal media can include, for example,one or more amino acids, one or more vitamins, one or more inorganicsalts, adenine sulfate, ATP, one or more trace elements, deoxyribose,ethanolamine, D-glucose, glutathione,N-[2-hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid] (HEPES), or oneor more other zwitterion buffers, hypoxanthine, linoleic acid, lipoidacid, insulin, phenol red, phosphoethanolamine, putrescine, sodiumpyruvate, thymidine, uracil, and xanthine. These ingredients arecommercially available.

Amino acid ingredients that can be included in the culture media of thedisclosure can include, for example, L-alanine, L-arginine,L-asparagine, L-aspartic acid, L-cystine, L-cysteine, L-glutamic acid,L-glutamine, glycine; L-histidine, L-isolcucine, L-leucine, L-lysine,L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine, and L-valine.

Vitamin ingredients that can be included in the media of the disclosurecan include, for example, ascorbic acid magnesium salt, biotin, cholinechloride; D-Ca⁺⁺ pantothenate, folic acid, i-inositol, menadione,niacinamide, nicotinic acid, paraminobenzoic acid (PABA), pyridoxal,pyridoxine, riboflavin, thiamine-HCl, vitamin A acetate, vitamin B₁₂ andvitamin D₂.

Inorganic salt ingredients that can be used in the media of thedisclosure can include, for example, CaCl₂, KCl, MgCl₂, MgSO₄, NaCl,NaHCO₃, Na₂HPO₄, NaH₂PO₄H₂O, and ferric citrate chelate or ferroussulfate chelate.

Trace elements that can be used in the media of the disclosure caninclude, for example, ions of barium, bromium, cobalt, iodine,manganese, chromium, copper, nickel, selenium, vanadium, titanium,germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin,zirconium, cadmium, zinc and aluminum. These ions can be provided, forexample, in trace element salts.

Additional ingredients that can optionally be included in the media are,for example, growth factors, insulin (especially as insulin-Zn⁺⁺) andtransferrin. These additional ingredients can be formulated into themedia at the typical biologic or physiologic concentrations. An ironsalt or chelate (e.g., ferric citrate chelate or ferrous sulfate) can beused in the media as a substitute for transferrin. Recombinant insulinor zinc based salts (e.g., ZnCl, etc.) can be substituted for animal- orhuman-derived insulin.

Many strategies have been employed to maximize the duration of cellculture, while maintaining the health of cells in culture. For example,frequent media changes, the introduction of nutrient-releasing materialsinto the media, perfusion culture and fed-batch processes have beenused.

In order to maximize cell culture nutrients in the media, and minimizewaste product build-up, media may be changed. Old media (which may bedepleted in nutrients and contain waste products) is removed and freshmedia is delivered to cells in culture. However, when media is changed,there is risk that the cell culture may be compromised. For example,when cell culture vessels are opened to remove old media and deliver newmedia, contamination may occur. Media changes also require the time andattention of cell culture professionals. That is, media changes havecontamination risk and labor costs.

Efforts have been made to extend the time between media changes, inorder to reduce labor costs and reduce the risk of contamination duringmedia changes. For example, cell culture media may contain anoverabundance of necessary nutrients, in order to ensure that sufficientnutrients are present in cell culture for a longer period of time, asthe cells use up the nutrients. This results in media which is overlyrich in certain nutrients such as, for example, glutamine or glucose.Glutamine (also referred to herein interchangeably as “L-glutamine”)hydrolyzes in an aqueous environment leading to the production ofammonium. Glutamine is also broken down by cells to produce ammonium.However, the production of ammonium from cellular metabolism ofglutamine may contribute less to an accumulation of ammonium in mediathan the hydrolysis of glutamine in an aqueous environment. Glucose isbroken down by cells form lactic acid. In a well-oxygenated cell culturesystem, the creation of lactic acid is a result of an overabundance ofglucose. Ammonium and lactic acid can build up and can be toxic to thecells. So, by providing media with an overabundance of these compounds,in an effort to provide sufficient nutrients to sustain cells in culturefor an extended period of time, it is paradoxically possible that cellsfind themselves in a less healthy environment than is necessary, astoxic by-products build up in the media over time.

Techniques have been developed to slowly release nutrients such asglucose into cell culture media by providing slow-release glucose. Forexample U.S. Pat. No. 3,926,723 discloses a method of culturing cellswhich includes providing a media which includes anenzymatically-releasable glucosyl moiety (starch) and an enzyme capableof releasing the glucosyl moiety (for example maltase and amylase) intocell culture media by enzymatically releasing glucose from the starchpolymer. Starch breaks down, in the presence of the enzymes, to formglucose.

Published U.S. Patent Application 2011/0244573 discloses pellets orpills which can added to cell culture, along with enzymes to releasenutrients from the structure of the pellet for fed-batch cultivation ofcell culture. Starch tablets with a lactose core were disclosed for E.coli cultivation. Lactose acted to degrade the starch of the tablet toprovide glucose to the cell culture.

Cells can also be grown in perfusion culture conditions. However,perfusion cell culture systems require significant equipment includingpumps, tubing and connectors, to ensure that fresh media is consistentlydelivered to the cells in culture, while maintaining sterility andavoiding unnecessary disruption of the cells. For example, internationalpatent publication WO2013/04044 entitled “Pre-Programmed Non-FeedbackControlled Continuous Feeding of Cell Cultures” discloses an apparatusfor providing continuous feed streams to a cell culture.

Other strategies include fed-batch or batch-fed processes that supplyfeed concentrates to cell cultures. However, fed-batch, or bolusfeeding, results in variability in the concentration of nutrients in acell culture over time, and can be labor intensive.

In static culture, products like Glutagro™, available from Corning,Incorporated, Corning, N.Y., or GlutaMAX™, available from LifeTechnologies, Carlesbad, Calif., are sometimes used. These products arecell culture media additives which contain glutamine dipeptides(L-alanyl-L-glutamine dipeptides) which slowly break down to releaseglutamine. This leads to much lower ammonium build up in the media incell culture, but it also releases alanine which causes some cell typesto perform poorly. Mammalian cell culture technologies are widely usedin biomedical research and pharmaceutical industries. Recombinantprotein production in mammalian cells is typically carried out usingsuspension-adapted cells for ease of scale up. Modern cell cultivationon a laboratory scale is mainly based on shaken cultures which aregenerally performed as batch cultures, i.e., nutrients are added at thestart of cultivation. Compared to industrial fed-batch processes, theshaken cultures are characterized by low volumetric cell and productyields. Shaken flasks provide only limited information for bioprocessdevelopment; culture parameters are often re-optimized in fed batchmodes and this significantly increases time and labor costs. In contrastto variable shaking cultures, in well controlled bioreactor scalecultivations, the fed-batch technology is mostly applied since itprovides better process control for nutrient and metaboliteconcentrations, oxygen level, biomass density, and media pH. Lack ofprocess control capabilities in batch cultures is the main reason thatprocess and media optimization results performed in batch mode cannot bedirectly translated into larger scale production in fed-batch operationmode. To overcome these issues miniature bioreactors or automationtechnologies (see for example, Legmann, R., et al., A predictivehigh-throughput scale-down model of monoclonal antibody production inCHO cells. Biotechnol Bioeng., 2009; 104; 1107-1120; Thomas, D., et al.,A novel automated approach to enabling high-thoughput cell linedevelopment, selection, and other cell culture tasks performed inErlenmeyer (shake) flasks, J. Assoc. Lab Autom., 2008; 13: 145-151;Gryseels, T., Considering cell culture automation in upstream bioprocessdevelopment, Bioproc. Intl., 2008; 6: 12-16), are used to allow highthroughput, fed-batch operation with automated feeding and control ofpH. Because these technologies are expensive, shaker flasks continue tobe widely used as the main scale down platform across industry andacademia in process development for mammalian cells (see Buhs, J.,Introduction to advantages and problems of shaken cultures, Biochem.Eng. J., 2001; 7: 91-98).

A major challenge is not only the adaptation of the fed-batch principlein the small scale, which is mostly done by intermittent feeding, butmore importantly reaching high cell densities at the same time. In batchprocess, cell density is determined by the concentration of growthlimiting nutrient source, media pH, and toxic metabolite concentrations.This creates a dilemma: high cell densities are only obtained whenenough glucose is available as carbon source. At the same time, bolusadditions of glucose can cause large transient increases in nutrientconcentration, which can lead to high osmolarity and high wastemetabolite concentrations, e.g., high glucose concentrations can lead toan increase in lactate production and pH decrease (see Zhou, W. C., etal., High viable cell concentration fed batch cultures of hybridomacells through online nutrient feeding, Biotechnol Bioeng., 1995; 46:579-587; Chee, F. W. D., et al., Impact of dynamic online fed-batchstrategies on metabolism, productivity an N-glycosylation quality in CHOcell cultures, Biotechnol. Bioeng., 2005; 89: 164-177). In addition,glycation of a recombinant antibody could be controlled by controllingglucose concentrations to low levels in the media (see Yuk, I. H., etal., Controlling glycation of recombinant antibody in fed-batch cellcultures, Biotechnol. Bioeng., 2011; 108; 2600-2610). The main pathwayof glucose utilization by the cells is glycolysis. Tumor derived celllines generally lose the ability to control glycolytic flux on the basisof energy needs (see Eigenbrodt, E., et al., New perspectives oncarbohydrate metabolism in tumor cells, R. Brietner (ed.), Regulation ofCarbohydrate Metabolism, Vol. 2., CRC Press, Boca Raton, Fla.). As aresult, glycolysis is controlled by the concentration of glucose in theextracellular growth medium. In a batch cell culture, glucose is oftenpresent at significantly higher concentrations (up to 50 mM) than foundin the blood stream (5 mM or less). It has been demonstrated thatdecreases in CHO (Chinese hamster ovary) cell viability is caused byhigh levels of toxic metabolite methylglyoxal, which is produced as aby-product of glycolysis at high glucose concentrations (see Chaplen, F.W. R., et al., Evidence of high levels of methylglyoxal in culturedChinese hamster ovary cells, Proc. Natl. Acad. Sci. USA, 1998;95:5533-5538; Roy, B. M., et al., Toxic concentrations of methylglyoxalin hybridoma cells culture, Cytotechnology, 2005; 46: 97-107).

To solve or mitigate these problems caused by high glucose concentrationin cell culture media there is a growing need for continuous substratedelivery source for high density cultivations. One example formicroorganism culture is EnBase™, an enzyme controlled glucose deliverysystem that was developed and is now commercially available to culturemicroorganisms (see Panula-Perala, J., et al., Enzyme controlled glucoseauto-delivery for high cell density cultivations in microplates andshake flasks, Microbial Cell factories, 2008; 7:31). EnBase™ usesglucoamylase/starch as a carbon source for the generation of glucose.Use of glucoamylase in cell culture media imposes limitations formammalian cells. Alternatively, glucose embedded intopolydimethylsiloxane resin has been used as a slow release technique forculture of H. polymorpha in shaker flasks (see Jeude, M., et al.,Fed-Batch Mode in Shake Flasks by Slow-Release Technique, Biotechnologyand Bioengineering, 2006; 95; commercially available as FeedBeads atkuhner.com). From the literature it is known that PDMS has highnonspecific binding capacity toward the proteins in cell culture mediaand as such is not desirable for mammalian cell culture applications.However, the use of a hydrogel based glucose delivery system has beendeveloped for the mammalian cell culture (see Hedge, S., et al.,Controlled release of nutrients to mammalian cell culture in shakeflasks, Biotechnol. Prog., 2012; 28: 1). This system is based onHEMA:EGDMA hydrogel disk with encapsulated glucose powder. Because ofunreacted residual monomers such system requires extensive washing ofhydrogels to mitigate cellular toxicity.

U.S. Pat. No. 8,563,066, to Sexton, et al., issued Oct. 22, 2013,entitled “Sustained Release of Nutrients In Vivo,” mentions nutritionalcompositions delivered in vivo in a time controlled manner sustainableover long periods of time, provide enhanced athletic performance,increased hand/eye coordination, and concentration on the task at hand.The compositions can include an aqueous suspension, comprising (a) oneor more nutritional supplements; and, (b) one or more hydrogelmicroparticles that encapsulate one or more nutritional supplements of(a), wherein the one or more hydrogel microparticles (i) have a diameterbetween 1 to 1000 micrometers; (ii) comprise one or more compounds thatare non-toxic, crosslinked, and that release the encapsulated one ormore nutritional supplements in a time controlled and sustained mannerin vivo; (iii) are pH-sensitive, wherein one or more compounds of thehydrogel microparticles do not swell at pH 1-3; and (iv) aretemperature-sensitive, wherein one or more compounds of the hydrogelmicroparticles have a lower critical solution temperature in aqueoussolution. The one or more compounds in (b)(ii) for the time controlledand sustained release of the nutritional supplements can bebiodegradable polymers, bioadhesives, binders, or a combination. Thebiodegradable polymers and binders can be one or more of poly(lactide)s,poly(glycolide)s, poly(lactide-co-glycolide)s, poly(lactic acid)s,poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s,polycaprolactone, polycarbonates, polyesteramides, polyanhydrides,poly(amino acids), polyorthoesters, polyacetyls, polycyanoacrylates,polyetheresters, poly(dioxanone)s, poly(alkylene alkylate)s, copolymersof polyethylene glycol and polyorthoester, biodegradable polyurethanes,polysaccharides and polysaccharide copolymers with polyethers. Sextonalso mentions that the microspheres can contain a mixture of nutritionalcompounds and the microsphere is composed of a biodegradable materialthat is released over a certain period of time. For example, in order toprovide an initial burst of nutrients to provide an immediate reservoirof energy or nutrients to the individual, the nutritional compounds areformulated as such and can contain a variety of carbohydrates, aminoacids, electrolytes, vitamins, etc. in differing ratios. The secondgroup can contain a differing ratio of carbohydrates:aminoacids:vitamins, etc., or strictly different or similar carbohydratesthat are released over a longer period of time to maintain a sustainablerelease of the nutrients. The formulation of the nutrients in themicrospheres and the timing of release can be varied depending on thetypes of activity, the individual, age, weight and nutritional needs.For example, a marathon runner (sustained nutrition over long period)would have different nutritional needs to a sprinter (burst ofnutrition).

As a general matter, products of biodegradation are generallyundesirable in in vitro or ex vivo cell culture. In embodiments, thematerial polymers selected for use in the present disclosure are notreadily biodegradable in situ (i.e., within and during the cellculture).

U.S. Pat. Publication 20090190135, entitled “Cell Culture Hydrogel WithpH Indicator,” mentions devices, compositions and methods formaintaining conditions in a cell culture and for measurement ofconditions in the cell culture. In particular, the invention provideshydrogel materials, apparatus and methods for several non-invasivetechniques of maintaining glucose and pH levels in cell cultures atnear-optimal levels and the non-invasive measurement of pH levels incell cultures.

This disclosure provides embodiments of a material to release glutamineand other nutrients, including glucose, in a steady, controlled manner,which allows for more robust cell culture conditions for longer periodsof time. In addition, this improved material creates conditions whichallow the media in cell culture to contain significantly less glucoseand glutamine, while still supporting superior cell growth and reducedammonium and lactate production. This allows for longer cell culturewithout requiring media changes (also known as “refeeding”) whilereducing the risk of contamination and increased labor costs thatfrequent media changes bring. Extending media's capacity to supportcells also may enable higher cell densities in vessels, which would alsomake cell culture more efficient.

FIGS. 1a and 1b are illustrations of a cell culture vessel 100 in anembodiment. The vessel (shown here as a flask, but the cell culturevessel may be any shape suitable for cell culture, including a petridish, a flask, a multi-well plate, a bag, a bioreactor, a multi-layercontainer, a multi-layer flask (as shown in FIG. 5 and FIG. 6, in anembodiment), a bed, a microcarrier (as shown in FIG. 2) or any othercontainer, vessel, flask, or surface suitable for cell culture. The cellculture vessel or surface may be suitable to support adherent ornon-adherent cells, animal cells, yeast cells, insect cells, eukaryoticcells, prokaryotic cells, tissue culture, organ culture, plant culture,primary cells, cell lines, genetically engineered cells or organisms, orany other cell culture purpose.

In the embodiment shown in FIGS. 1a and 1 b, the vessel 100 has a bottom109, a top 110, side walls 112, and a necked opening 131, shown in FIG.1a with a cap 130 attached to the necked opening. The inside of thevessel shown in FIG. 1a is a cell culture chamber 111. FIG. 1b is across-section of the cell culture vessel shown in FIG. 1 a, taken atplane A-A shown in FIG. 1 a. As shown in FIG. 1 b, the vessel 100 hasinterior surfaces. The top wall 110 has an interior surface 115, alsocalled a ceiling 115. The bottom wall 109 has an interior surface 113,also called a bed 113. Each side wall 112 has an interior surface 132.

FIG. 2 is a microcarrier embodiment. FIG. 2 shows a microcarrer 200 incross-section. The microcarrier has a microcarrier body 149 which has amicrocarrier surface 150. Material 151 is shown in a layer on themicrocarrier surface 150.

FIG. 3a-e are illustrations of cross-sections of a cell culture vessel,taken at plane A-A shown in FIG. 1 b, showing embodiments of material oninterior surfaces of a cell culture vessel. In each of FIGS. 3a-3e , across-section of the cell culture vessel 100 in the embodiment shown inFIGS. 1a and 1b is shown. The cell culture vessel 100 has a top wall 110having an interior top surface 115 or ceiling 115, side walls 112 havinginterior side wall surfaces 132, a bottom wall 109 having an interiorbottom surface 113 or bed 113 and a cell culture chamber 111. In FIG. 3a, material 151 is shown on the interior surface 113 or bed 113 of bottomwall 109. In FIG. 3b , material 151 is shown on the interior surface 115of top wall 110, or ceiling 115. In FIG. 3c , material 151 is shown onthe interior surface 132 of side wall 112. In FIG. 3d , material 151 isshown on the interior surface 132 of the opposite side wall 112. In FIG.3e , material 151 is shown on the interior surfaces 115, 113, and 112 ofeach of top wall 110, bottom wall 109 and side walls 112. Although FIG.3e shows material on each of the interior surfaces of the cell culturechamber 115, 113, and 112 of each of top wall 110, bottom wall 109 andside walls 112, material may be present on one, more than one, but lessthan all, or all of the interior surfaces 115, 113, and 112 of each oftop wall 110, bottom wall 109 and side walls 112.

In embodiments, material 151 may completely cover a surface, or maypartially cover a surface. In embodiments, material 151 is a coatingapplied to one or more of interior surfaces 115, 113, and 112 of each oftop wall 110, bottom wall 109 and side walls 112. A coating may beapplied with wet chemistry by methods such as cast and cure. That is, asolution of material may be introduced into vessel 100 and allowed tocoat one or more of internal surfaces of vessel. Additional steps may betaken, such as a curing step or a drying step, to allow the coating toaffix to one or more of internal surfaces of vessel. In embodiments,material is a film applied to one or more of interior surfaces 115, 113,and 112 of each of top wall 110, bottom wall 109 and side walls 112. Afilm may be applied to one or more of internal surfaces of vessel by anymethod known in the art, including applying a film to one or more ofinternal surfaces prior to assembling vessel. In embodiments, materialis integral with one or more of the top wall 110, bottom wall 109 andside walls 112. For example, material may be extruded to form one ormore of top wall 110, bottom wall 109 and side walls 112.

FIGS. 4a and 4b are images of embodiments of cell culture mediaextending materials 151, in the absence (FIG. 4a ) and in the presence(FIG. 4b ) of cell culture media. Shown in FIG. 4a is a photograph of anembodiment of the cell culture extending material, apoly(ethylene-co-vinyl acetate) “EVA” material (40% vinyl acetate) with20% glucose, solvent cast onto a surface. In dry conditions, glucosecrystals 300 are visible under a tight layer of polymer 301. As shown inFIG. 4a , glucose crystals are sequestered in the polymer. Inembodiments, the polymer, together with its trapped or sequesterednutrients (in the case of FIGS. 4a and 4b , glucose) are cell cultureextending materials 151. After incubating the material in aqueous dPBSsolution, which is osmotically similar to media, for 96 hours, thematerial has changed, as shown in FIG. 4b . Without being limited bytheory, it is hypothesized that glucose, trapped in the polymer, hasabsorbed water, creating pressurized pockets 315 in the polymer 301leading to pumping of the nutrients out of the polymer, into thesurrounding solution. This can be described as an osmotic pump, wherethe nutrients are driven out of the polymer pocket into the media by theconcentration gradient between the pocket and the media.

In embodiments of the present invention, a multi-layer flask isprovided. An embodiment of the multi-layer flask 100 of the presentinvention is illustrated in the partial cut-away perspective view shownin FIG. 5. The multi-layer flask 100 has an outer vessel body 101defined by a top wall 110, a bottom tray (not shown), sidewalls 112, andend walls 114. Disposed within the flask 100 are individual cell growthchambers 111 as can be seen more clearly in FIG. 6. The individual cellgrowth chambers 111 are each defined by a bottom surface or bed 113 anda top surface or ceiling 115. The surfaces 113 and 115 are attached tothe flask body 101 along sidewalls 112 and end walls 114. Inembodiments, at least one bottom surface 113 within each chamber 111 isa gas permeable, liquid impermeable material capable of providing asurface for the growth of cells 117. The gas permeable, liquidimpermeable material may provide the surface upon which cells attach, orthe bed of the cell growth chamber, or it may be the opposite surface,or the ceiling of the cell growth chamber. The bottom surface 113, orthe cell culture surface 113 may be flexible or rigid. Each top surface115, in embodiments, is a rigid, generally gas impermeable material thatwill provide support to the cell growth chamber 111 and the multi-layercell culture vessel. The surfaces of the multi-layer flask may be clear,opaque, colored or colorless. In an embodiment of the present invention,there are tracheal spaces 118 between each cell growth chamber 111. Theopposing top surface 115 of the chamber 111 defines an upper wall orceiling to the cell growth chamber 111 as well as a bottom portion of atracheal chamber 118. The tracheal chamber 118 is therefore inclusive ofa gas permeable, liquid impermeable surface 113 of a first cell growthchamber and an opposing surface 115 of a second growth chamber 111.Supports 119 may also be present to provide structural support tointegrally incorporate the surfaces 113 and 115 in forming growthchambers 111 in alternation with tracheal air spaces 118 within theunitary flask 101. Each cell growth chamber 111 therefore alternateswith a tracheal chamber 118 in vertical successive orientation.

In an embodiment of the present invention, the individual cell growthchambers 111 permit cellular growth on gas permeable membranes 113 suchthat multiple cell growth chambers 111 are integral with the body 101 ofthe multi-layer flask 100 and are capable of being completely filledwith nutrient media for the growth of cells. In embodiments, these cellgrowth chambers are approximately 2 mm in height. The series of trachealair spaces 118 through the multi-layer flask 100 provide gaseouscommunication between the cells 117 growing on gas permeable surfaces113, in media 127 in the individual cell growth chambers 111 inside themulti-layer flask, and the external environment. The tracheal spaces 118allow oxygenation of media located within cell growth chambers 111through the gas permeable surfaces 113. Further, the tracheal chambers118 may take the form of any air gap or space, and do not allow entranceof liquid. As a result, a rigid cell culture multi-layer flask 100having multiple growth chambers 111, alternating with tracheal spaces118, is cooperatively constructed to afford the benefit of equivalentgaseous distribution to a large volume of cells 117. In embodiments, themulti-layer flask may have ports 120, which may have plugs or portcovers 121. In addition, the multi-layer flask may be of any shape. Forexample, the multi-layer flask may have corners 107.

FIG. 6 is a cross-section of a cell culture vessel 100, taken at planeB-B shown in FIG. 5, showing embodiments 1-5 of the material on interiorsurfaces of the multi-layer cell culture vessel embodiment shown in FIG.5. In cell culture chamber 1 (111), a top wall 110 has an interiorsurface or ceiling 115, and a layer of cell culture media extendingmaterial 151 on a bottom surface or bed 113. In embodiments, the bed 113is made from liquid impermeable, gas permeable film. A tracheal space118 is below the bed 113, supported by supports 119. In embodiment (1)of the multi-layer cell culture chamber, the cell culture mediaextending material is on the bottom surface or bed 113.

In cell culture chamber 2, cell culture media extending material is onthe interior surface or ceiling 115 of top wall (which in this secondlayer of the multi-layer is also the layer which provides supports 119to support gas permeable, liquid impermeable film and form trachealspace 118). In cell culture chambers 3 and 4, material 151 is on sidewalls. In cell culture chamber 5, cell culture media extending materialis on all interior surfaces of the cell culture chamber 111. Those ofordinary skill will understand that cell culture media extendingmaterial may be provided on any surface in a cell culture vessel, in theconfigurations illustrated in FIGS. 1, 2, 3, 5, 6, 7, 8 or any otherconfiguration.

FIG. 7 is an illustration of a method of using the material, in a singlelayer cell culture vessel embodiment. In FIG. 7, cell culture mediaextending material 151 is on the bottom wall or bed 113 of a cellculture vessel. Cells 159 sit on top of the bed 113, covered with media160. Nutrients 161 are provided by media, but are also released fromcell culture media extending material 151, as shown by arrows in FIG. 7.In addition, water from media 160 enters material 151, as shown by thearrow in FIG. 7. Water may move into the polymer film from the culturemedia. While not being limited by theories about the mechanism of actionof the release of nutrients into media, it may be that water hydratessolid nutrient particles, forming a saturated solution in pockets withinthe polymer, as shown in FIG. 4b . This solution volume creates pressurewithin the film which drives the solvated nutrient, which may be, forexample, glutamine or glucose, through the polymer and out into theculture media at a steady rate (see FIGS. 14-18). That steady ratemaintains a low useful concentration of glutamine to support the cellsin culture while keeping ammonium production rates low.

FIGS. 8a-8d are illustrations of embodiments of methods of using fourembodiments of the material in a multi-layer flask environment. In FIG.8a , material 151 is on ceiling 115 of a cell culture chamber in amulti-layer cell culture vessel. Cells 159 sit on top of the bed 113,which may be a liquid impermeable, gas permeable film, covered withmedia 160. Nutrients 161 are provided by media, but are also releasedfrom material 151, as shown by arrows. In addition, water from media 160enters material 151, (not shown in FIG. 8a , but see FIG. 7, and alsoFIGS. 4a and 4b ). In FIG. 8b , material 151 is on the bottom wall orbed 113 of a cell culture chamber of a multi-layer cell culture vessel.Cells 159 sit on top of the bed 113, covered with media 160. Nutrients161 are provided by media, but are also released from material 151, asshown by arrows. In FIG. 8c , material 151 is on the interior surfaces132 of side walls 112 of a cell culture chamber of a multi-layer cellculture vessel. Cells 159 sit on top of the bed 113, covered with media160. Nutrients 161 are provided by media, but are also released frommaterial 151, as shown by arrows. In FIG. 8d , material 151 is on all ofthe interior surfaces of a cell culture chamber of a multi-layer cellculture vessel. Cells 159 sit on top of the material 151, on bed 113,covered with media 160. Nutrients 161 are provided by media, but arealso released from material 151, as shown by arrows.

In some cell culture vessels, media is provided to the cell culturechamber in a way that provides a layer of air inside the vessel. In thisway, oxygen levels are maintained at a health level as oxygen diffusesinto the media at the media-air interface. In multi-layer cell culturedevices, such as those shown in FIGS. 5 and 6, media fully fills thecell culture chambers. In these embodiments, oxygen is provided to cellsin culture by diffusion across a gas permeable liquid impermeablemembrane which forms the bed of the cell culture chamber 113. The bed113 has contact with media on the cell culture chamber side and with airon the tracheal space 118 side of the membrane.

In embodiments, material 151 is provided on the ceiling surface 115 ofthe cell culture chambers in multi-layer cell culture vessels. In thisway, oxygen can diffuse into the cell culture chamber through the gaspermeable liquid impermeable membrane of the bed 113 of the cell culturechamber and nutrients can pass into the media via material 151 presenton the ceiling of the cell culture chamber.

Examples of cell culture materials for nutrient release include polymerscontaining sequestered nutrient particles. Suitable polymer materialallows water to slowly permeate the polymer material, releasing nutrientparticles captured in the polymer matrix. The water is then able tobegin dissolving the nutrient particles, freeing the nutrient to bereleased through the polymer and out of the media. This release ofnutrient from the polymer material can act as in response to an osmoticpump, allowing for steady release of glutamine over days.

Polymers useful for the current invention need to be able to containnutrients. This can be done by mixing the nutrient with polymermaterials prior to forming the polymer material into a surface incontact with cell culture media. Nutrients may be in crystal form, andcan be captured by or bound in a polymer layer, as shown in FIGS. 4a and4b . Or, this can be done by providing a layer of polymer between alayer of nutrient and the cell culture media, allowing the nutrients tobe released through the polymer layer over time. Or, the nutrients canbe trapped between layers of polymer. Nutrients that are trapped beneatha layer of polymer or trapped between layers of polymer are also“sequestered”. And, the polymer must be able to release the nutrient ata controlled rate. That is, if the polymer is too porous, nutrients willbe released too quickly, on the order of hours. To extend the life ofcell culture, nutrients need to be released over a period of days oreven weeks. If the polymer is impermeable to water, no nutrients will bereleased for the benefit of the cell culture. In embodiments, thepolymer has characteristics that allow for the release of nutrients atan appropriate rate. One way to describe this characteristic is in thepolymer's hygroscopic characteristics. Hygroscopic polymers have anability to attract and hold water molecules (adsorb water molecules)from the surrounding environment. Examples of hygroscopic polymersinclude poly(ethylene-co-vinyl acetate), poly(ethyl cellulose), PDMS,poly(ether/amide) copolymers such as PEBAX 2533, and others. Hygroscopicpolymers can be distinguished from hydrogels. Hydrogel is defined as “apolymeric material that exhibits the ability to swell and retain asignificant fraction of water within its structure, but will notdissolve in water(http://www.sciencedirect.com/science/article/pii/S2090123213000969). Ahydrogel is defined by IUPAC as “a gel in which the swelling agent iswater” (Notes: 1. The network component of a hydrogel is usually apolymer network. 2. A hydrogel in which the network component is acolloidal network may be referred to as an aquagel.) In general,hydrogels swell in water, and, in water, contain a high percentage ofwater. For purposes of this disclosure, a hydrogel is a polymer materialthat swells to more than 50% water by weight in an aqueous environment.In embodiments, the hygroscopic material disclosed herein are polymersthat swell to less than 50% water by weight in an aqueous environment.The water-absorbing characteristics and water-releasing characteristicsof the polymer determine the rate at which the polymer is able torelease nutrients, dissolved in water, over time. Nutrients diffuse outof or through the polymer material.

In embodiments the media extending materials are coatings or films,applied to a cell culture surface, which are capable of releasing cellculture nutrients over time. In embodiments the media extendingmaterials disclosed herein are polymers which swell to less than 50%water by weight in an aqueous environment, which are mixed withnutrients, or have captured or sequestered nutrients, or containnutrients. In embodiments, nutrients are captured in or sequestered inpolymer in crystalline or solid form.

In additional embodiments, the disclosure provides methods of culturingcells in the presence of a cell culture surface having media extendingmaterials where the nutrients, captured in a polymer matrix, dissolve inthe presence of aqueous media, and are slowly released into the media.When the nutrients dissolve in an aqueous environment, an osmotic pumpmay be created in pockets in the polymer matrix, allowing nutrients toflow from the more concentrated environment of the polymer pocket, intothe media. This sequestration of nutrients in the polymer matrix allowsfor the slow introduction of nutrients into media without requiringmedia changes. This sequestration also allows cells to be cultured, instatic culture or in dynamic culture conditions, in the presence oflower concentrations of nutrients which, in higher concentrations, maybe toxic to cells, directly or indirectly.

The rate of diffusion of nutrients out of the polymer over time is alsoaffected by the thickness of the film or layers of film, the nature ofthe nutrients, the diffusion gradient between the polymer and the media,temperature, and other factors.

These polymer/nutrient materials can be made in any form including cast,extruded, molded, or provided as coatings and thin films to provide highsurface area for exchange. Also, once films are extruded or made, filmscan be molded, over-molded, insert-molded or otherwise applied to asurface exposed to cell culture. The material itself can be molded orover-molded to form a surface exposed to cell culture. The material canform at least a part of a surface of a cell culture compartment. Thematerial can form part of a surface that is also the surface upon whichcells attach and grow (“cell culture surfaces”), or they may form partof surfaces that are not cell culture surfaces. Or, the material canform at least a part of a microcarrier or other surface used to culturecells.

In embodiments, the material is extruded to form a cell culture vessel.In additional embodiments, the material is formed as a coating on asurface of a cell culture vessel. In additional embodiments, the coatingmay be a film. The coating may be cast on a surface of a cell culturevessel, or the coating may be formed as a film that is later applied asa film to a cell culture surface. Films can be molded, over-molded,insert-molded or otherwise applied to a surface exposed to cell culture.The formed film may be cast, extruded, or molded, or formed by any othermethod known in the art. Solvent casting can be used to generate filmsand coatings.

In additional embodiments, the material is applied in more than onelayer to a cell culture surface. For example, a material containing anutrient can be applied to a surface to form a first layer, and then asecond material, which does not contain a nutrient, can be applied ontop of the first layer to form a second layer. The application of asecond layer which does not contain a nutrient can be applied to slowthe release of one or more nutrients from the first layer, as thatnutrient has to move out of the first layer, and also through the secondlayer, before becoming accessible to media (see, for example, Film 19 inTable 1 below).

EXAMPLES

The following examples illustrate the preparation of exemplary materialsfor enhancing long term cell culture conditions.

Example 1 Formulation of Materials

Film 13 was cast from a 10% solution of poly(ethylene-co-vinyl acetate)(40% VA) in methylene chloride was blended with 20% glucose powder(milled sample) by polymer weight and 20% glutamine by polymer weight.This solution was knifed onto 0.010″ thick polystyrene film to a 0.010″thickness which dried to approximately 0.002″ thickness. This resultingfilm was then coated with a 10% solution of the same polymer (EVA)without nutrients added. The final film thickness was 0.013″ includingthe 0.010″ polystyrene base film. Film 15 was made the same way but with20% glucose and 30% glutamine. For layer 4 of film 19, the “overcoat”,polymer was dissolved in solvent without the nutrient to form anovercoat. Film 19 was made with an additional basal layer. The firstbase layer had 40% glucose in EVA followed by another layer with 30%glucose, followed by a layer with 20% glucose and 25% glutamine,followed by the overcoat of EVA without nutrients.

TABLE 1 L- Film Polymer Glucose glutamine Film 13 10% solution of 20%20% poly(ethylene-co-vinyl acetate) (40% VA) in methylene chloride Film15 10% solution of 20% 30% poly(ethylene-co-vinyl acetate) (40% VA) inmethylene chloride Film 19 10% solution of Layer 1: 40% glucose in 25%in multi- poly(ethylene-co-vinyl EVA layer 3 layer acetate) (40% VA) inLayer 2: 30% glucose in material methylene chloride EVA Layer 3: 20%glucose and 25% glutamine in EVA Layer 4: (overcoat) EVA without glucoseor gluta- mine

Example 2 Formation of Material Films

The films profiled in FIGS. 12-18 (discussed below) were solvent castonto cell culture surfaces. Solvent casting is a process of dissolving apolymer in a suitable solvent. In this example, a suitable solvent is asolvent which dissolves the polymer but not the nutrient. Then thepolymer solution is knifed onto a film and allowed to dry. This producesa composite film material having a polymer containing a nutrient. Solid(undissolved) nutrients were present in the film in clumps. That is,because the nutrients were not dissolved in the cast material, they wereunevenly distributed throughout the composite polymer material (seeFIGS. 4a and 4b ). Solvent casting can be used to generate films andcoatings. Thermal processing may also be used to provide films ofmaterial 151. These films could be inserted into a mold and overmoldedwith a first polymer layer of the film facing the mold cavity so as tocreate a weld when the mold is filled with a polymer compatible with thefirst polymer layer. The osmotic pump side of the film would then befacing the cell culture chamber. The film could also be welded in asecondary operation.

FIG. 9 is a graph showing glucose consumption vs. ammonium production inthe presence of traditional media having a 4 mM L-glutamineconcentration (and a 1 g/L glucose concentration) compared toexperimental low glutamine media conditions having a 0.5 mM L-glutamineconcentration (and a 1 g/L glucose concentration). FIG. 9 illustratesthat in low glutamine media conditions, ammonium production is reducedover the 96 hours of culture shown in the graph of FIG. 9. FIG. 9illustrates that at the end of a 96 hour culture, ammonium production innormal glutamine conditions is approximately twice that measured fromcultures in low glutamine conditions. In addition, glucoseconcentrations fell off faster in low glutamine concentrationconditions. Increasing levels of ammonia (NH₄) are correlated withdecreasingly healthy cell culture conditions. FIG. 9 illustrates that,in the presence of low glutamine, cell culture conditions remainhealthier longer. Glucose concentrations fell off faster in the presenceof low glutamine concentrations because cell populations increasedcompared to the normal glutamine conditions (see FIG. 10). Statedanother way, cell populations grew up faster under low glutamineconditions than under normal glutamine conditions. This may be due toreduced ammonium build-up (as shown in FIG. 11).

FIG. 10 is a graph illustrating CHOk1 cells harvested after 96 hoursgrowing in traditional media having a 4 mM L-glutamine concentration(and a 1 g/L glucose concentration) compared to experimental lowglutamine media conditions having a 0.5 mM L-glutamine concentration(and a 1 g/L glucose concentration). As shown in FIG. 10, the flasksformulated with low glutamine had an increased yield of cells comparedto cells in the presence of normal glutamine conditions. This data, incombination with the graphs shown in FIG. 9 indicate that cells growfaster in low glutamine conditions. This matches reports of batchculture conditions that found cells grew faster in low glutamine/lowammonia culture conditions (Low-Glutamine Fed-Batch Cultures of 293-HEKSerum-Free Suspension Cells for Adenovirus Production Lee, Y. Y. et al.,Biotechnol. Prog. 2003, 19, 501-509). This group also measured higherviral titer in cells grown in low glutamine/low ammonia conditions. Thisdisclosure shows that in fed-batch cell culture conditions, providingglutamine in batches in low concentrations is favorable.

For the purposes of this disclosure “low glutamine concentration” meansa concentration below 2 mM L-glutamine, below 1.5 mM L-glutamine orbelow 1.0 L-glutamine. Cells need some glutamine in culture, but notvery much. That is, L-glutamine concentrations of below 2 mML-glutamine, below 1.5 mM L-glutamine or below 1.0 L-glutamine aresufficient for supporting cell culture. However, because glutaminebreaks down in culture, the levels of glutamine provided in media usedfor static culture is usually above 2 mM.

FIG. 11 is a graph illustrating ammonium production per cell per dayafter 96 hours growing in traditional media having a 4 mM L-glutamineconcentration (and a 1 g/L glucose concentration) compared toexperimental low glutamine media conditions having a 0.5 mM L-glutamineconcentration (and a 1 g/L glucose concentration). Ammonia produced percell per day (cumulative ammonium produced divided by IVCD, integratedviable cell density) demonstrated an 80% reduction as compared to thestandard concentration L-glutamine control (set 2).

FIG. 12 is a graph illustrating glucose release in mg/cm² from glucoseand glutamine-containing embodiments of the material over time. Exampleof 3 polymer/nutrient film formulations that contains both glucose andglutamine were tested for glucose release. Examples 13, 15 and 19 aredefined in Table 1. FIG. 12 plots the release rate of glucose to supportcell macro-nutrient requirements and demonstrates that relative changesin the film formulation can result in a wide range of nutrient releaserates.

FIG. 13 is a graph illustrating glutamine release in mg/cm² from glucoseand glutamine-containing embodiments of the material over time. Exampleof 3 polymer/nutrient film formulations that contains both glucose andglutamine (See Table 1 for material compositions). FIG. 13 plots therelease rate of L-glutamine to support cell macro-nutrient requirementsand demonstrates that relative changes in the film formulation canresult in a wide range of nutrient release rates. Depending on thesystem requirements the formulation can be adjusted to match demand.

FIG. 14 is a graph illustrating glucose release in g/L fromglucose-containing embodiments of a material over time in hours. FIG. 14illustrates glucose release from a PEBAX™ 2533 material containing 10%glucose, and having an additional top coating layer of PEBAX™ withoutglucose of about 0.001″ in thickness. PEBAX™ is a polyamide/polyetherblock copolymer available from Arkema, King of Prussia, Pa. Material wassolvent cast onto cell culture surfaces and incubated in aqueous dPBSsolution, which is osmotically similar to media. The concentration ofglucose in g/L was measured at time increments. The measuredconcentration of glucose was multiplied by 3.5 to give equivalentconcentration of the released glucose when it enters a 2 mm media headheight, resulting in a measurement of average glucose release over timeof 0.5 g/L for a 2 mm media height. This media head height is the mediaspace in a multi-layer cell culture flask, the HYPERStack®, availablefrom Corning Incorporated, Corning, N.Y., illustrated in FIG. 5. ThePEBAX™ material released glucose at a steady rate of over a 72 hourperiod of 0.5 g/L.

FIG. 15 is a graph illustrating glucose release in g/L from anotherglucose-containing embodiment of the material over time. FIG. 15illustrates glucose release from EVA (ethyl vinyl acetate, 40% weight %VA) containing 10% glucose, and having an additional top coating layerof EVA without glucose of about 0.001″ in thickness. EVA material wassolvent cast onto cell culture surfaces and incubated in aqueous dPBSsolution, which is osmotically similar to media. The concentration ofglucose in g/L was measured at time increments. The measuredconcentration of glucose was multiplied by 3.5 to give equivalentconcentration of the released glucose when it enters a 2 mm media headheight. EVA materials containing glucose released glucose at a steadyrate over a 96 hour period 1.5 g/L.

FIG. 16 and FIG. 17 are graphs illustrating glucose release in g/L froma glucose-containing ethyl cellulose embodiment of the material overtime. FIG. 15 illustrates glucose release from EC300 grade ethylcellulose (available from Sigma, Dorset, UK, cat #247499) materialcontaining 30% glucose, and FIG. 16 illustrates glucose release fromEC100 grade ethyl cellulose (available from Sigma, Dorset, UK, cat#200654,) material containing 50% glucose, each having an additional topcoating of polymer without glucose of about 0.001″ in thickness. In bothcases, material was solvent cast onto cell culture surfaces andincubated in aqueous dPBS solution, which is osmotically similar tomedia. The concentration of glucose in g/L was measured at timeincrements. The measured concentration of glucose was multiplied by 3.5to give equivalent concentration of the released glucose when it entersa 2 mm media head height. Both ethyl cellulose materials containingglucose released glucose at a steady rate over a 120 hour period. Thecalculated glucose release from the EC300 material having 30% glucosewas 0.7 g/L and the calculated glucose release from the EC100 materialhaving 50% glucose was 2.8 g/L.

FIG. 18 is an additional graph illustrating glucose release in g/L fromanother glucose-containing embodiment of the material over time. FIG. 18illustrates glucose release from EVA (ethyl vinyl acetate, 40% weight %VA) containing 30% glucose and having an additional top coating layer ofEVA without glucose of about 0.001″ in thickness. EVA material wassolvent cast onto cell culture surfaces and incubated in aqueous dPBSsolution, which is osmotically similar to media. The concentration ofglucose in g/L was measured at time increments. The measuredconcentration of glucose was multiplied by 3.5 to give equivalentconcentration of the released glucose when it enters a 2 mm media headheight. EVA materials containing glucose released glucose at a steadyrate over a 96 hour period of 4.9 g/L.

The disclosure has been described with reference to various specificembodiments and techniques. However, it should be understood that manyvariations and modifications are possible while remaining within thescope of the disclosure.

1. A cell culture vessel having a surface comprising a cell culturemedia extending material.
 2. The cell culture vessel of claim 1 whereinthe cell culture media extending material comprises a polymer comprisingat least one sequestered nutrient.
 3. The cell culture vessel of claim 1wherein the cell culture media extending material comprises ahygroscopic polymer and at least one nutrient.
 4. The cell culturevessel of claim 3 wherein the hygroscopic polymer comprises EVA.
 5. Thecell culture vessel of claim 1 wherein the cell culture media extendingmaterial comprises at least one coating.
 6. The cell culture vessel ofclaim 1 wherein the cell culture media extending material comprises afilm.
 7. The cell culture vessel of claim 1 wherein the vessel comprisesat least one cell culture compartment, each cell culture compartmentcomprising a bed, a ceiling and at least one wall.
 8. The cell culturevessel of claim 7 wherein the bed comprises the cell culture mediaextending material.
 9. The cell culture vessel of claim 7 wherein theceiling comprises the cell culture media extending material.
 10. Thecell culture vessel of claim 7 wherein the at least one wall comprisesthe cell culture media extending material.
 11. The cell culture vesselof claim 7 wherein at least two of the bed, the ceiling and the at leastone wall comprise the cell culture media extending material.
 12. A cellculture media extending material comprising a hygroscopic polymer and atleast one nutrient.
 13. The cell culture media extending material ofclaim 12 wherein the material forms part of a cell culture vessel. 14.The cell culture media extending material of claim 12 wherein thematerial forms at least a part of a microcarrier.
 15. The cell culturemedia extending material of claim 12 wherein the hygroscopic polymercomprises EVA.
 16. The cell culture media extending material of claim 12wherein the at least one nutrient comprises glucose.
 17. The cellculture media extending material of claim 12 wherein the at least onenutrient comprises glutamine.
 18. The cell culture media extendingmaterial of claim 12 wherein the material is a film.
 19. The cellculture media extending material of claim 13 wherein the material is afilm and wherein the film is on the ceiling of a cell culture chamber.20. A method of culturing cells comprising: (a) introducing cells into acell culture chamber comprising a cell culture media extending material,wherein the cell culture media extending material comprises ahygroscopic polymer comprising at least one sequestered nutrient; (b)incubating the cells in the cell culture chamber in the presence ofmedia having an L-glutamine concentration less than 2 mM.
 21. The methodof claim 20 wherein the L-glutamine concentration is between 0 and 1.5mM.
 22. The cell culture vessel of claim 1 comprising: at least one cellculture compartment; and media with an L-glutamine concentration lessthan 2 mM within the at least one cell culture compartment.
 23. The cellculture vessel of claim 22, wherein the L-glutamine concentration isbetween 0 and 1.5 mM.